Treprostinil reduces endothelial damage in murine sinusoidal obstruction syndrome
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Sinusoidal obstruction syndrome (SOS) is a major complication after hematopoietic stem cell transplantation and belongs to a group of diseases increasingly identified as transplant-related systemic endothelial disease. Administration of defibrotide affords some protection against SOS, but the effect is modest. Hence, there is unmet medical need justifying the preclinical search for alternative approaches. Prostaglandins exert protective actions on endothelial cells of various vascular beds. Here, we explored the therapeutic potential of the prostacyclin analog treprostinil to prevent SOS. Treprostinil acts via stimulation of IP, EP2, and EP4 receptors, which we detected in murine liver sinusoidal endothelial cells (LSECs). Busulfan-induced cell death was reduced when pretreated with treprostinil in vitro. In a murine in vivo model of SOS, concomitantly administered treprostinil caused lower liver weight-to-body weight ratios indicating liver protection. Histopathological changes were scored to assess damage to liver sinusoidal endothelial cells, to hepatocytes, and to the incipient fibrotic reaction. Treprostinil indeed reduced sinusoidal endothelial cell injury, but this did not translate into reduced liver cell necrosis or fibrosis. In summary, our observations provide evidence for a beneficial effect of treprostinil on damage to LSECs but unexpectedly treprostinil was revealed as a double-edged sword in SOS.
Murine liver sinusoidal endothelial cells (LSECs) express prostanoid receptors.
Treprostinil reduces busulfan-induced cell death in vitro.
Treprostinil lowers liver weight-to-body weight ratios in mice.
Treprostinil positively affects LSECs in mice but not hepatic necrosis/fibrosis.
KeywordsProstacyclin VOD SOS HSCT Defibrotide Transplant-related toxicity
Sinusoidal obstruction syndrome
Hematopoietic stem cell transplantation
Liver sinusoidal endothelial cells
Cyclic adenosine monophosphate
Allogeneic stem cell transplantation
18S ribosomal RNA
White blood cell
Red blood cell
Liver weight to body weight
Sinusoidal obstruction syndrome (SOS), previously referred to as veno-occlusive disease (VOD), is a severe hepatic complication that occurs after hematopoietic stem cell transplantation (HSCT). Initial damage of liver sinusoidal endothelial cells (LSECs) caused by the combination of high-dose cytotoxic drug therapy/irradiation and inflammatory mediators released in an allogeneic immune reaction are central to the pathophysiology of the disease [1, 2]. The course and severity of SOS are highly variable and difficult to predict: mild to moderate SOS typically resolves within weeks. In contrast, severe SOS can progress to multi-organ failure rendering SOS a life-threatening orphan disease .
Defibrotide is the only drug approved for the treatment of SOS [4, 5]. Its mechanism of action is enigmatic: originally, defibrotide was shown to stimulate the production of prostacyclin/PGI2 in different vascular beds [6, 7] and to act as an agonist at A1- and A2A-adenosine receptors , accounting for some of the beneficial actions . Stimulation of Gs-coupled receptors, such as the A2A-adenosine receptor and the β2-adrenergic receptor, increases the proliferation and survival of endothelial cells [10, 11]. The cognate receptor of prostacyclin/PGI2 is also a Gs-coupled receptor. Inflammatory cytokines and other mediators reduce endothelial prostacyclin/PGI2 production . Conversely, activation of the I prostanoid (IP) receptor and possibly of other Gs-coupled E prostanoid receptors (EP2, EP4) elicits protective actions . In addition, IP receptor stimulation counteracts fibrotic stimuli [13, 14]. In fact, when prophylactically administered by continuous intravenous infusion, PGE1 halved the incidence of SOS after allogeneic bone marrow transplantation . However, an independent trial failed to replicate the beneficial effect . In contrast to PGE1, treprostinil is selective for Gs-coupled receptors with a preference of IP receptors . Treprostinil was approved for the treatment of pulmonary hypertension. Accordingly, the clinical experience with treprostinil covers the sum of several thousand patient years: treprostinil is reasonably well tolerated and its human pharmacology is well understood [18, 19, 20, 21].
Here, we explored the hypothesis that treprostinil interfered with the cascade triggering and/or sustaining damage to LSECs in a murine model of SOS. We examined the action of treprostinil in mice subjected to allogeneic hematopoietic stem cell transplantation. The observations provided evidence for a beneficial effect of treprostinil on damage to LSECs, but this failed to translate into precluding the development of SOS.
Materials and methods
Isolation of primary murine cells and experiments with transformed murine LSECs
Primary hepatocytes and LSECs were isolated by liver perfusion as described previously . Isolated primary hepatocytes and LSECs were immediately homogenized for RNA extraction. Vijay H. Shah (Mayo Clinic and Foundation, Rochester, MN, USA) kindly provided transformed murine sinusoidal endothelial cells (TSECs) with stable expression of SV40 large T-antigen . TSECs were cultured in endothelial cell medium containing 5% fetal bovine serum, 1% penicillin/streptomycin, and 1% endothelial cell growth supplement (ECGS) (ScienCell Research Laboratories, San Diego, CA). For cell viability experiments, 24 h after plating (5 × 103 cells/24-well plate), 10 μM treprostinil was given to the medium. After 1 h pretreatment with treprostinil, busulfan (Sigma-Aldrich, Vienna, Austria) was added at concentrations ranging from 10 μM to 1 mM. Cell viability was assessed after 48 h. For this purpose, cells were washed twice with PBS, trypsinized, and counted using a hemocytometer and trypan blue. For MTT cell metabolic activity experiments, 24 h after plating (1.5 × 103 cells/96-well plate) in phenol-red free medium, 10 μM treprostinil was given to the medium. After 1 h pretreatment with treprostinil, busulfan was added at concentrations ranging from 125 μM to 1 mM. MTT assay was assessed after 48 h according to manufacturer’s instructions and absorbance was measured at 570 nm (Thermo Fisher Scientific, Waltham, MA). For [3H]cAMP accumulation assays, TSECs were incubated for 16 h with medium containing [3H]adenine (1 μCi ml−1) and samples were processed as described previously [24, 25, 26]. Treprostinil (Remodulin®) was kindly provided by SciPharm SàRL (2540 Luxembourg City, Luxembourg).
Analysis of gene expression by quantitative PCR
Primer sequences used for qPCR analysis
Murine gene symbol
Male BALB/c and C57BL/6J mice were either purchased from the Jackson Laboratory (Bar Harbor, ME) through Charles River Germany (Sulzfeld, Germany) or were bred in-house (C57BL/6J). Mice were between 8 and 10 weeks old, and their body weight was in the range of 20–25 g. Animal housing and husbandry were in accordance with the recommendations and requirements defined by the Federation of Laboratory Animal Science Associations (FELASA) in Europe. Mice were kept on a 12-h light-dark cycle in isolated ventilated cages with ≤ 5 mice/cage (Smart Flow and Easy Flow; Tecniplast, Buguggiate, Italy) at 21 ± 3 °C. Mice were fed autoclaved standard laboratory chow (commercial control diet for mice; Ssniff R/M-H, Soest, Germany) and water ad libitum. Animal technicians monitored animal welfare and health status daily under the supervision of a veterinarian. The experimental protocol was reviewed by the animal ethics committee of the Veterinary University of Vienna, approved by the Austrian Ministry of Science and Research under licenses BMWFW-68.205/0103-WF/V/3b/2015 and BMWFW-68.205/0047-V/3b/2018 and conducted according to the guidelines of FELASA and ARRIVE. Predefined humane end points included emaciation (i.e., weight loss > 25%), loss of activity (reduced mobility, prolonged crouching), loss of grooming/ruffled fur, or labored breathing. Mice meeting these criteria were killed by cervical dislocation.
Experimental animal model
The murine model of SOS described by Zeng et al. [27, 28] is based on an allogeneic hematopoietic stem cell transplantation (allo-HSCT), where preconditioning is achieved by whole body irradiation. In that model, the maximum of SOS is observed 15 days after allogeneic hematopoietic stem cell transplantation. After adaptation for 7 days, BALB/c mice were randomly divided into three groups: healthy control, allo-HSCT, and allo-HSCT + treprostinil. Mice assigned to allo-HSCT + treprostinil were pretreated 1 day before HSCT with treprostinil subcutaneously (0.15 mg kg−1 8 h−1) as described previously . On the same day, recipient mice, i.e., the groups allo-HSCT and allo-HSCT + treprostinil, were exposed to whole body irradiation (7.5 Gy, split doses, 2 Gy min−1; Siemens Primus, 6MV, Siemens Austria): mice were placed in single chambers of an irradiation pie with 15 mice per pie. The radiation dose delivered was verified with a dosimeter. On the next day, recipient mice received 5 × 106 bone marrow mononuclear cells containing 1.14 ± 0.26% hematopoietic stem cells via tail vein injection. Bone marrow mononuclear cells were obtained from donor C57BL/6 mice. Mice belonging to the group allo-HSCT + treprostinil were daily treated with subcutaneous injections of treprostinil for 15 days. Thereafter, all mice (healthy control, allo-HSCT, allo-HSCT + treprostinil) were euthanized.
Histology and immunohistochemistry
On day 15 after HSCT, mice were weighed and livers were immediately removed after euthanasia, weighed, fixed in 4% formalin, and embedded in paraffin for subsequent slicing. Liver weight was normalized for body weight by calculating the liver weight/body weight ratio. Sections prepared from formalin-fixed, paraffin-embedded organ specimens were stained with hematoxylin-eosin (H&E) or with Masson-Goldner trichrome using standard protocols. Light microscopic images were captured with a PixeLINK camera and the corresponding acquisition software on a Zeiss Imager Z.1.
Scoring of histopathology
Histological sections (two sections/animal) stained with H&E and Masson-Goldner trichrome were evaluated by a board-certified pathologist (JH), who was blinded to the nature of the treatment. The scoring system was based on the system described by Qiao and Zeng et al. [27, 28]. Histological changes were reviewed and scored (0 to 3 points/item) for the following seven pathological features: (i) endothelium injury in liver sinusoid or small hepatic veins, (ii) subendothelial hemorrhage, (iii) internal hemorrhage in the hepatic sinusoid, (iv) liver cell necrosis, (v) fibrosis of the central veins, (vi) hepatic sinusoidal fibrosis, and (vii) inflammation in the central veins.
Blood sampling and evaluation of plasma parameters
Blood of euthanized mice was collected by heart puncture for analysis of blood cell count and biochemical parameters: white blood cell (WBC), red blood cell (RBC), and platelet (PLT) counts were determined using the Vet animal blood counter (scil animal care, Viernheim, Germany). Plasma levels of bilirubin and alanine aminotransferase (ALT) were assessed using the test strip-based Reflotron Plus analyzer (Roche, Basel, Switzerland).
The primary outcome parameter was hepatomegaly, and the number of animals was based on the following assumptions: the liver-to-body weight ratio (liver weight as % of body weight) was to increase from a mean of 5.5% with a standard deviation of 0.5% to 7.5 ± 0.7% on day 15 . This increase was to be halved by treprostinil. Based on this assumption, we calculated that 18 animals/group were required to detect a statistically significant difference of p < 0.05 with a 90% probability. We included one and two additional mice in the treprostinil-treated and in the control recipient group, respectively, to account for possible dropouts due to bone marrow transplant failure; treprostinil enhances bone marrow transplantation , hence we assumed that the dropout rate would be smaller. Secondary outcomes included ALT, bilirubin, and pathohistological scores.
Target receptors of treprostinil are expressed by murine LSECs
Treprostinil attenuates busulfan-induced cell death in TSECs
Pretreatment with treprostinil reduces hepatomegaly
Histopathological consequences of treprostinil treatment
SOS is a challenging disease from the perspective of both basic research and clinical management. Toxic injury to liver sinusoidal cells is thought to be the central pathogenic event that occurs in the early phases after HSCT. We employed a murine SOS model, which is known to peak on day 15 and to subsequently resolve spontaneously . Accordingly, we selected day 15 to assess the extent of liver damage. Our observations recapitulated the original findings; this is most readily evident by considering the extent of hepatomegaly and the elevations of ALT levels: our values are in excellent agreement with those reported by Zeng et al. . Similarly and consistent with the observations, we also found that changes in bilirubin levels were less useful to monitor hepatic damage. This is not unexpected because ablation of the bone marrow and the subsequent reconstitution of the bone marrow by hematopoietic stem cell transplantation are likely to affect red blood cell dynamics and thus the production of bilirubin. Our study was powered to detect a beneficial effect of treprostinil provided that it resulted in a reduction in liver weight. Based on hepatomegaly as primary outcome parameter, the treatment of recipient mice with treprostinil was indeed effective in mitigating SOS.
Our working hypothesis posited that treprostinil acted via Gs-coupled prostanoid receptors on the LSECs. We verified the presence of transcripts of Ep2, Ep4, and Ip receptors in LSECs. Similarly, the histological analysis suggested that treprostinil reduced injury to liver sinusoidal endothelial cells. The effect, which we detected in the histopathology, was modest, presumably because our analysis was limited to a snapshot on day 15, i.e., long after the initial injury. More importantly, the histological analysis revealed a damaging effect of treprostinil on the hepatocytes. This is surprising, because prostaglandins are thought to be beneficial in liver injury and their involvement in regeneration is complex: PGE2 and to a lesser extent PGI2 stimulate hepatocyte proliferation ; the effect is relevant under in vivo conditions, because genetic ablation of the prostaglandin degrading enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH) or its inhibition by a small molecule enhances liver regeneration after partial hepatectomy . Conversely, liver regeneration after surgical removal of liver tissue is blunted upon inhibition of cyclooxygenase-2 and to a lesser extent of cycloxygenase-1, the enzymes that catalyze the conversion of arachidonic acid to prostaglandin H2, the precursor of PGE2, PGI2, and other prostaglandins . These findings suggest a role of endogenous PGE2 in promoting hepatocyte regeneration. It is less clear which prostanoid receptors mediate the beneficial actions of PGE2: originally, Gq-coupled E prostanoid receptors were shown to be more important for stimulating the proliferation of hepatocytes than Gs-coupled receptors , but in ischemia-reperfusion injury, the protective effect was conveyed by stimulation of the EP4 receptor . However, more recently, the beneficial effects of PGE2 and in particular of the EP4 receptor have been questioned : in fact, inhibition of PGE2 synthesis—by deletion of the inducible microsomal PGE synthase-1 or by its inhibition with a small molecule—mitigated liver cell necrosis resulting from ischemia and reperfusion. Similarly, blockage of the EP4 receptor—not of the EP1 or of the EP2 receptor—protected against hepatocyte damage and reduced necrotic areas in mice, in which the EP4 receptor was absent due to genetic deletion . Thus, the available evidence shows that the activation of Gs-coupled prostaglandin receptors results in both enhanced and reduced hepatocyte necrosis depending on the nature of the injury and the time of the snapshot. In fact, the conflicting results of Kuzumoto et al.  and of Nishizawa et al.  can be rationalized by considering that the time points sampled differed: Kuzumoto et al.  examined early events (i.e., 2 h after reperfusion), while the observation period of Nishizawa et al.  focused on changes occurring days after ischemia and reperfusion. The pathophysiology underlying damage caused by SOS is presumably more akin to that of ischemia and reperfusion than that of partial hepatectomy. Furthermore, our analysis focused on late events, which are presumably more relevant for the clinical treatment of SOS. Taken together, the previously published findings and our current observations indicate that treprostinil is a double-edged sword. While its action on the LSECs and on platelets may be beneficial, its action on macrophages/Kupffer cells and/or hepatocytes limits the usefulness in the treatment of SOS. This is presumably also true for other agonists, which target Gs-coupled prostanoid receptors.
Defibrotide is the only drug which is currently available for the treatment of SOS [4, 5]. The approval of defibrotide is based, in part, on a phase III trial, which—for ethical reasons—relied on a carefully selected historical control group . Based on this trial, it is clear that the unmet medical need is large: the numbers needed to treat were 5 and 6 for preventing death within 100 days and 6 for a complete response (i.e., a reduction in serum bilirubin < 2 mg/100 mL, creatinine clearance > 80% of initial value, and oxygen saturation > 90% with ambient air), respectively. It is evident that there is room for improvement. In contrast to treprostinil, which is a chemically defined small molecule, defibrotide is a polydisperse mixture of oligonucleotides obtained from porcine intestinal mucosa. The heterogeneity of defibrotide hampers progress, because it is not clear which of its many reported actions are relevant to improve the outcome in SOS. Administration of defibrotide is associated with a substantial risk of hemorrhage (i.e., affecting 5 to 10% of the patients) and of hypotension. Finally, data on the long-term safety of defibrotide are not yet available. The advantage of treprostinil is not only its well-defined mechanism of action but also the fact that its safety profile is well understood in both adults [18, 19, 20] and children [18, 19, 20]. However, treprostinil can only be considered a viable candidate drug for SOS, if it shows convincing efficacy in a credible preclinical model.
We stress that we used a dose of 0.45 mg kg−1 day−1 treprostinil, which—after correction for allometry—corresponds to the clinical dose in man: it is the dose which can be safely administered in the treatment of pulmonary hypertension and shows maximum efficacy [18, 19, 20]. In fact, this dose provided the optimum beneficial effect in promoting hematopoietic stem cell transplantation in lethally irradiated recipient mice . In previous work, we noted a bell-shaped dose-response curve of treprostinil, suggesting that a higher dose of treprostinil is less beneficial than the currently used dose. We cannot formally exclude that a variation in treprostinil dose would have an uncovered window, where the beneficial effect would have outweighed the detrimental action. However, we consider it unlikely that this can be detected with an ethically justifiable number of experimental animals because of the large inherent variability in the course of SOS. Currently, prostaglandins such as PGE1 are not recommended for the treatment of SOS , because an encouraging initial trial  was not replicated . At the very least, our observations provide an explanation for this failure: in our murine SOS model, stimulation of Gs-coupled was protective for the endothelium of the liver sinusoids, but we also detected a damaging effect of treprostinil on hepatocytes. This detrimental action is likely to limit or cancel out any benefit arising from the administration of prostaglandins in human SOS.
Open access funding provided by Medical University of Vienna. The authors thank Ursula J. Lemberger, Christoph H. Oesterreicher, Melitta Penz, and Marion Mussbacher for experimental advice, reagents, and helpful discussions. The authors further thank Katrin Meissl, Michaela Prchal-Murphy and Veronika Sexl for the excellent collaboration at the Veterinary University of Vienna.
This work was supported by SciPharm with the project number FA641A0113.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
The experimental protocol was reviewed by the animal ethics committee of the Veterinary University of Vienna, approved by the Austrian Ministry of Science and Research under licenses BMWFW-68.205/0103-WF/V/3b/2015 and BMWFW-68.205/0047-V/3b/2018 and conducted according to the guidelines of FELASA and ARRIVE.
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